Process for preparing purified s-bel and r-bel and compositions thereof

ABSTRACT

A process for resolution of racemic BEL into its individual enantiomeric constituents by chiral HPLC. A method for determining the role of specific isoforms of iPLA 2  in biologic processes.

This application claims the benefit of priority of U.S. ProvisionalPatent Application 60/365,903 filed Mar. 19, 2002 which is incorporatedin its entirety by reference.

FIELD OF THE INVENTION

This invention relates to a process for the preparation ofenantiomerically pure and optically enriched S-BEL from a compositioncomprising enantiomers S-BEL and R-BEL using HPLC chiral chromatography.More particularly, this invention relates to a process for preparingoptically active purified S-BEL and a composition comprising purifiedS-BEL. This invention also relates to a process for preparing purifiedR-BEL and a composition comprising purified R-BEL. This invention alsorelates to a process for selective inhibition of iPLA₂ lipases. Moreparticularly this invention relates to a process for selectiveinhibition of lipases which perform functions in cells.

BACKGROUND OF THE INVENTION

Calcium-independent phospholipases A2 (iPLA₂s) constitute an importantgroup of intracellular enzymes which function to hydrolyze esterifiedfatty acids from membrane phospholipids in response to agoniststimulation, changes in intracellular calcium ion homeostasis, andalterations in cellular energy requirements (for reviews, see 1-3). Inearly studies, we and others demonstrated that the majority of PLA₂activity in most non-circulating mammalian cell types including smoothmuscle cells (4), pancreatic jpβ-cells (5,6), cardiomyocytes (7,8), andhippocampal neurons (9) was calcium-independent and inhibited by racemic(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one(rac-BEL). Based upon activity assays, calcium requirements, loss ofarachidonylated phospholipid mass, and inhibition of iPLA₂ by rac-BEL(R-BEL), a diverse array of cellular processes has been proposed to beregulated by iPLA₂s, including arachidonic acid (AA) release (10-15),cellular proliferation (16), assembly of VLDL (17), putinergicreceptor-stimulated kallikrein secretion (18), apoptosis (19),endothelial cell PAF synthesis (20), and induction of iNOS and nitricoxide production (21).

Three distinct subclasses of iPLA₂ have been identified at the geneticlevel (with subsequent confirmation of iPLA₂ catalytic activity byrecombinant technologies) and have been designated iPLA₂α, iPLA₂β, andiPLA₂γ, in order of their discovery (22-24). The iPLA₂s have beencategorized based upon their strict conservation of nucleotide-binding(GXGXXG) and lipase (GXSTG) consensus sequences (FIG. 1). Two of theiPLA₂ subclasses, iPLA₂β and iPLA₂γ, have been cloned from mammaliancDNA libraries while the ortholog of iPLA₂α (patatin), at the time ofthis writing (with 98.7% of tire human genome sequenced), has not beenidentified in mammals. Calcium-independent phospholipase A₂β containseight ankyrin-repeat domains which are believed to facilitateintracellular sorting (23, 25, 26) and a CaM-binding domain near theC-terminus which binds calcium-activated CaM and regulates enzymeactivity (27) (FIG. 1). The binding of CaM to iPLA₂β results ininhibition of iPLA₂β activity which is reversible through removal ofCa⁺² and subsequent dissociation of CaM from the C-terminus of iPLA₂β(27, 28). In this paradigm, iPLA₂β is regulated through alterations incellular calcium ion homeostasis and becomes activated afterdissociation from its complex with Ca⁺²/CaM when intracellular calciumstores are depleted by SERCA inhibitors, calcium-ionophores, or agoniststimulation (29, 30). In contrast, the recently identified iPLA₂γ doesnot bind CaM and its mechanisms of regulation are unknown at present.

Studies of iPLA₂ have utilized the mechanism-based suicide inhibitorrac-BEL as a pharmacologic tool to identify the type of intracellularphospholipase A₂ involved in many diverse cellular processes. Sincerac-BEL inhibits both iPLA₂β and iPLA₂γ at low microflora concentrations(24, 25, 31, 32), it is impossible to assign rac-BEL-mediated inhibitionof AA release to iPLA₂β or iPLA₂γ activities. Accordingly, it becamenecessary to develop pharmacologic approaches which could discriminatebetween iPLA₂β and iPLA₂γ to facilitate identification of their biologicroles. In addition, it has been reported in the Journal (33, 34) thathigh concentrations of BEL (25 μM) partially inhibit themagnesium-dependent cytozoic phosphatidate phosphohydrolase, PAP-1,which converts phosphatidic acid to diacylglycerol (DAG). In thoseinvestigations, it was proposed that PAP-1 inhibition by BEL wouldprevent activation of protein kinase C leading to attenuated AA release.However, “rescue” experiments in which PKC was exogenously activated byphorbol esters or diacyl glycerol analogs after BEL treatment were notreported by the authors to address their hypothesis (33, 34).

BRIEF DESCRIPTION OF INVENTION

In an aspect, a process for preparing S-BEL and R-BEL comprises passinga racemic mixture comprising S-BEL and R-BEL through at least onechromatographic column packed with an optical resolution packingmaterial to optically resolve S-BEL from the racemic mixture. In anaspect the packing material comprises a silica.

In an aspect a method of separating a racemic composition comprisingR-BEL and S-BEL into purified R-BEL and S-BEL respectively comprisespassing a racemic composition comprising R-BEL and S-BEL through an HPLCchiral separation column which comprises a stationary phase and elutingsaid R-BEL and said S-BEL from said column.

In an aspect a process for chromatographically resolvingenantiomerically pure or optically enriched BEL from a mixturecontaining two enantiomers of BEL using chiral chromatography comprisesa liquid mobile phase and a chiral stationary phase.

In an aspect the solid chiral stationary phase is attached to a silicaor silica.

In an aspect, the inventors have shown the identification of iPLA₂β andnot iPLA₂γ, as the Mediator of AVP-induced Arachidonic Acid Release inA-10 Smooth Muscle Cells by enantioselective Mechanism-BasedDiscrimination of intracellular lipases.

In an aspect, an isolated and purified S-BEL.

In an aspect, an isolated and purified R-BEL.

In an aspect, a purified composition comprising R-BEL.

In an aspect, a purified composition comprising S-BEL.

In an aspect, a method of identifying whether a lipase enzyme ismetabolically active in a cellular environment comprises contacting saidcellular environment with at least one of S-BEL and R-BEL anddetermining the identity of the lipase based on the interaction of saidlipase with at least one of S-BEL and R-BEL.

In an aspect, a diagnostic method for determining the metabolic activityof a lipase in a cellular environment comprises contacting said cellularenvironment with at least one of S-BEL and R-BEL and determining theidentity of the lipase based on the interaction of said lipase with atleast one of S-BEL and R-BEL.

In an aspect, a diagnostic kit/method for determining the metabolicactivity of a lipase in a cellular environment comprises providing acellular environment comprising a lipase for which it is desired todetermine the metabolic activity, contacting said cellular environmentwith at least one of S-BEL and R-BEL and determining the identify and/oractivity of the lipase based on the interaction of said lipase with atleast one of S-BEL and R-BEL.

In an aspect, a method of identifying whether a lipase enzyme ismetabolically active in a cellular environment comprises contacting saidcellular environment with at least one of S-BEL and R-BEL anddetermining the identify of the lipase based on the interaction of saidlipase with S-BEL.

In an aspect, a method of identifying whether a lipase enzyme ismetabolically active in a cellular environment comprises contacting saidcellular environment with at least one of S-BEL and R-BEL anddetermining the identity of the lipase based on the interaction of saidlipase with R-BEL.

In an aspect, a method of inactivating a lipase enzyme which ismetabolically active in a cellular environment comprises contacting saidcellular environment with at least one of S-BEL and R-BEL and therebyrendering the lipase inactive.

In an aspect, a method of selectively inhibiting iPLA2γ in a compositioncomprises effectively contacting the same with an effective inhibitingamount of S-BEL.

In an aspect, a method for pharmacologically distinguishing iPLA2γ fromiPLA2β comprises contacting a candidate iPLA2 with S-BEL and if theselectivity is high of S-BEL to the candidate iPLA₂, then determiningthat the iPLA2 is iPLA2γ. As used herein, the term “candidate” includesmembers of the iPLA2 family.

In an aspect, a method of differentially inhibiting iPLA2γ and iPLA2βcomprises contacting the same with S-BEL, observing selectivity of S-BELtoward iPLA2γ or iPLAαβ and determining that said iPLA2β has beeninhibited. In an aspect, iPLA₂β is selectively inhibited.

In an aspect, a method for identifying/determining whether iPLA₂β oriPLA₂γ is active in a metabolic pathway or chemical process believed toutilize iPLA₂ enzymes comprises contacting said pathway or process withat least one of R-BEL and S-BEL, determining if said R-BEL or S-BEL hasan inhibiting effect and further determining whether this effect is oniPLA₂γ or iPLA₂β, identifying iPLA₂β or iPLA₂γ as active.

In an aspect a S-BEL inhibited iPLA₂γ is provided.

In another aspect an R-BEL inhibited iPLA₂γ is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the calcium-independent phospholipase A₂ (iPLA₂) genefamily and sequence alignment of iPLA2 nucleotide and lipase consensusmotifs.

FIG. 2 depicts separation of BEL enantiomers by chiral HPLC.

FIG. 3 depicts inhibition of α-chymotrypsin by racemic, (R)-and (S)-BEL.

FIG. 4 depicts selective inhibition of iPLA₂β and iPLA₂γ by racemic,(R)-, and (S)-BEL.

FIG. 5 depicts inhibition of AVP-mediated arachidonic acid liberation inA-10 smooth muscle cells.

FIG. 6 depicts inability of racemic BEL to inhibit A-10 smooth musclecell cytosolic (PAP-1) and membrane-bound (PAP-2) phosphatidatephosphohydrolase activities.

FIG. 7 depicts inability of phorbol-12-myristate-13-acetate and1,2-dioctanoyl-sn-glycerol to reconstitute arachidonic acid liberationin A-10 cells treated with (S)-BEL.

FIG. 8 depicts translocation of PKCδ and PKCε in A-10 smooth musclecells is unaffected by pre-treatment with (S)-BEL

FIG. 9 depicts racemic BEL, (R)-BEL, or (S)-BEL do not inhibitAVP-induced MAPK phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect, the inventors employed rac-BEL to demonstrate a 1000 foldselectivity iPLA₂ vs. cPLA₂ and SPLA₂ family members (31, 32). In afurther aspect, based upon the increasing appreciation of the utility ofchiral pharmacologic agents in enhancing the specificity of inhibitorstoward targeted biologic processes, we discovered that (R)- and (S)-BELdifferentially inhibits iPLA₂β and iPLA₂γ activities. Moreover, wediscovered that development of chiral mechanism-based inhibitorsprovides an increased degree of discrimination between specific targetedenzyme systems and those of “non-specific inhibition. We exploited theresolution of racemic BEL into its individual enantiomeric constituentsby chiral HPLC. We showed the selective inhibition of iPLA₂β by (S)-BELand iPLA₂γ by (R)-BEL, and demonstrated that BEL-mediated inhibition ofAA release in A-10 cells is likely mediated by iPLA₂β and not due toinhibition of iPLA_(2γ) or the effects of BEL on MAPK or PKC activation.

Agonist-stimulated release of arachidonic acid (AA) from cellularphospholipids in many cell types (e.g. myocytes, β-cells, and neurons)has been demonstrated to be primarily mediated by calcium-independentphospholipases A2 (iPLA₂s) which are inhibited by the mechanism-basedinhibitor(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one(BEL).

As used herein, the terms “preparative HPLC” and “HPLC” include a HPLC(high performance liquid chromatography) process for the isolation andpurification of compounds including compounds such as S-BEL and R-BEL.(see www.pharm.uky.edu/ASRG/HPLC/hplcmytry.html)

In an aspect, chemical separation is brought about by utilizing ourdiscovery that S-BEL and R-BEL have different migration rates given aselected chiral chromatographic column and mobile phase employedtherewith.

The separation of enantiomers such as S-BEL and R-BEL by means of liquidchromatography (LC) using chiral stationary phases is based on thereversible diasteromeric association between the chiral environment inthe column and the enantiomers fed to the column.

In an aspect the column is a racemic resolveable and separable column.

Useful illustrative compositions to be separated and refined hereininclude racemic mixtures, including those comprising racemates. In anaspect a racemate is fed to a chiral chromatographic column producingone, two or multiple eluents respectively therefrom copying R-BEL andS-BEL respectively.

In an aspect a continuous chiral chromatrographic column is employed. Inan aspect a batch or semi-continuous type chiral chromatrographic columnis employed. In an aspect, the column is operably and suitablyeffectively configured to provide the desired separation, purificationand elution.

As used herein, the term “purification” includes a process of separationor extracting a target or a designated compound(s) such an opticalisomer(s) from other related compounds such as a related optical isomer,wherein each compound has a characteristic peak under chiralchromatographic conditions.

As used herein, the term “racemic” denotes the presence of equimolar ornearly equimolar amounts of dextrorotatory and levorotatory enantiomersof a compound.

In an aspect S-BEL comprisesS—(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one andR-BEL comprisesR—(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one.

In an aspect a racemic composition of S-BEL and R-BEL is separated,refined or made nonracemic by using a suitable chiral chromatograph.

In an aspect a suitable chiral chromatograph is one which is enantiomerseparable capable, i.e., can separate enantiomers.

In an aspect a racemic composition of BEL entantiomers are resolvedproviding one or multiple eluents comprising S-BEL and R-BELrespectively. In an aspect a racemic BEL composition is fed to a HPLCchiral chromatograph column to provide a chiral chromatograph producteluent comprising chiral chromatographic produced R-BEL and S-BEL

In an aspect BEL entantiomers are resolved by HPLC utilizing a Chirexcolumn of 3,5 dinitrobenzoyl-R-phenylglycine attached to a silica matrixas the stationary chiral phase.

In an aspect the column is calibrated.

In an aspect, the chiral column is equilibrated withhexane/dichloroethane/ethanol and optical enantiomers are elutedisocratically at a flow rate of 2 ml/min.

In an aspect isocratic elution is carried out in a process whereby thechromatographic procedure or separation in which the composition of theeluent is maintained constant or nearly constant.

In an aspect, elution of BEL compositions from the column is monitoredby UV absorbance at 280 nm. Peaks corresponding to the R BEL and S BELenantiomers were collected, dried under nitrogen and stored at −20° C.

In general the degree of separation or refinement is greater than about60% (racemic) to about 90% and in the range from about 70% to about 85%.

Illustrative useful chiral HPLC columns include those which operate byimmobilizing single enantiomers onto a stationary phase(s) in thecolumn(s). Achieving capable and sufficient resolution depends to anextent on the effective formation of transient diastereoisomers on thesurface of the column packing which in an aspect is the immobile phase.Generally the compound which forms the more stable diastereoisomer willbe most or preferentially retained whereas the opposite enantiomer willform a less stable diastereoisomer will elute first. It is desired inthis discovery to achieve a high degree of discrimination betweenenantiomers in passing through and effectively contacting the phases ofthe chiral HPLC chromatographic column to isolate and purified S-BEL andR-BEL. Useful illustrative operably configured chiral HPLC columnsinclude those which operate effectively on the basis of chirality whichis topical handedness or the property of nonidentity of an object withits mirror image.

In an aspect, the chromatography comprises a liquid mobile phasecomprises 3,5-dinitrobenzoyl-(R)-phenylglycine attached to a silicamatrix as a solid chiral stationary phase.

In an aspect, the eleutant comprises at least one of a purified andisolated S-BEL, a purified and isolated R-BEL, a composition comprisingS-BEL and a composition comprising R-BEL.

As used herein, the terms “silica” and “silica based matrix” include anyuseful silicon oxide or silicon dioxide including silica gel, silicaoxide, which is a colourless or white solid effectively used as thesupport or basis for chromatographic procedures. Generally in silicachromatographic processes, the silica is heated to above about 100C todrive off water. In an aspect silica material serves as a support.

Illustrative useful chiral stationary phases for use in chiralchromatography include those materials selected from the groupconsisting of Type I, II, III, IV and V CSP (chromatographic stationaryphase) as classified by Irving Wainter (57).

Illustrative useful Type I CSP include those which differentiateenantiomers by formation of complexes based on attractive interactionswhich include hydrogen bonds, p-p interactions and dipole stacking.

Illustrative useful Type II CSPs include those which involve acombination of interactions and inclusion complexes to produce aseparation, these are largely based on cellulose derivatives.

Illustrative useful Type III CSPs include those which rely on soluteentering into chiral cavities to form inclusion complexes, such as thecyclodextrin type of column developed by Prof. D. W. Armstrong (58).Other useful CSPs include crown ethers and helical polymers such aspolytriphenylmethyl methacrylate.

Illustrative useful Type IV CSP include those which separate by means ofdiastereomeric metal complexes which are known as Chiral ligand exchangechromatrograph (CLEC) and developed by Davankov (59, 60).

Illustrative useful chiral columns are also classified by chemical type.Illustrative useful chiral stationary phases include Brush type(Pirkle), cellulose, cyclcodextrin, macrocylic antibiotics, protein,ligand exchange and crown ethers.

These illustrative useful and other useful illustrative chiral columnsinclude those listed in the Online Guide to Chiral HPLC by Mark Earl ,see www.raell.demon.co.uk/chem/CHIbook/Chiral.htm.

A useful comprehensive guide to chiral HPLC applications is disclosed atwww.chromtech.se/chiral.htm.

In an aspect this discovery is employed to provide an enhanced improvedseparation process for racemic compositions of S-BEL and R-BEL andprovides a straightforward expeditious one step low cost method. Thisdiscovery is useful as a research tool to identify the role of lipasesin biologic processes, and to probe active sites as by proteonomics.This discovery is also useful for proof of concept of specificisoformers used in lipase studies.

Recently, the family of mammalian iPLA₂s has been extended to includeiPLA₂γ which previously could not be pharmacologically distinguishedfrom iPLA₂β.

To determine if iPLA₂β or iPLA₂γ (or both) were the enzes responsiblefor arginine vasopressin (AVP)-induced AA release from A-10 cells, itbecame necessary to selectively inhibit iPLA₂β and iPLA₂γ in intactcells. Racemic BEL was separated into its enantiomeric constituents bychiral HPLC. Remarkably, (S)-BEL was approximately an order of magnitudemore selective for iPLA₂β in comparison to iPLA₂γ. Conversely, (R)-BELwas approximately an order of magnitude more selective for iPLA₂γ thaniPLA₂β. The AVP-induced liberation of AA from A-10 cells was selectivelyinhibited by (S)-BEL (IC₅₀ about 1 μM but not (R)-BEL, demonstratingthat the overwhelming majority of AA release is due to iPLA₂β and notiPLA₂γ activity. Furthermore, pre-treatment of A-10 cells with (S)-BELdid not prevent AVP-induced MAPK phosphorylation or PKC translocation.Finally, two different cell-permeable protein kinase C activators(phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) couldnot restore the ability of A-10 cells to release AA after exposure to(S)-BEL, thus supporting the downstream role of iPLA₂β in AVP-induced AArelease.

This discovery has multiple utilities and illustratively is useful forproof of concept of specific isoformers used in lipase studies, fortagging iPLA₂ family members and labelling molecules. In an aspect thisdiscovery is useful as one or more research tools, diagnostic method(s)and tool for selective inhibition of iPLA₂ family members such astargeted disease states, mode of action studies and investigativemethods.

The following examples are described in detail in order to facilitate aclear understanding of the invention. It should be understood, however,that the detailed expositions of the application of the invention, aregiven by way of illustration only and are not to be construed aslimiting the invention since various changes and modifications withinthe spirit of the invention will become apparent to those skilled in theart from this detailed description.

EXAMPLES:

Experimental Procedures

Materials—BEL, phorbol myristate 13-acetate (PMA), and1,2-Dioctanoyl-sn-glycerol (DOG) were obtained from Calbiochem. A ChirexHPLC column comprised of a stationary phase of (R)-phenylglycine linkedthrough an amide linkage to 3,5-dinitrobenzoic acid was purchased fromPhenomenex. Three-fold-crystallized chymotrypsin,N-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin, fatty acid freebovine serum albumin (low endotoxin), antibodies against PKCα, and mostother reagents were obtained from Sigma. AVP was purchased from Bachem.HPLC-grade organic solvents and channeled LK6D Silica Gel 60 Å thinlayer chromatography plates (Whatman) were obtained from FisherScientific. Enhanced chemiluminescence (ECL) reagents and film werepurchased from Amersham PharmaciaL-α-1-palmitoyl-2-[1-¹⁴C]-arachidonyl-phosphatidylcholine,L-α-1-palmitoyl-2-[1-¹⁴C]-oleoyl-phosphatidylcholine,[5,6,8,9,11,12,14,15-³H(N)]-arachidonic acid, and L-α-dipalmitoyl[glycerol-¹⁴C(U)]-phosphatidic acid were purchased from NEN. A-10 cellsderived from rat aortic smooth muscle were obtained from the ATCC andcultured as described previously (10). (R) and (S) enantiomers of(E)-6-(iodomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one(αNpI6) were received (Univ. of Illinois, Urbana). Anti-Active MAPK(pTEpY) and Anti ERK ½ antibodies were obtained from Promega. Antibodiesagainst PKCε and PKCδ were received (St. Louis Univ., St. Louis).Antibodies against PKC₁ were obtained from BD Transduction Laboratories.

Recombinant iPLA₂ Enzymes—Recombinant iPLA₂β was expressed and purifiedfrom Sf9 cells as previously described (35). Recombinant full-lengthiPLA₂γ was expressed in Sf9 cells and the membrane fraction was washedand isolated as previously described (24).

Separation of BEL Enantiomers—BEL enantiomers were resolved by HPLCutilizing a Chirex column of 3,5-dinitrobenzoyl-(R)-phenylglycineattached to a silica matrix as a stationary chiral phase. The chiralcolumn was equilibrated with hexane/dichloroethane/ethanol (150/15/1)and optical enantiomers were eluted isocratically at a flow rate of 2ml/min. Elution of BEL compositions from the column was monitored by UVabsorbance at 280 nm. Peaks corresponding to the (R)- and(S)-enantiomers were collected, dried under N₂, and stored at M-1-20° C.The concentration of BEL for each experiment was determinedspectrophotometrically based on UV absorbance (ε₂₈₀=6130 cm⁻¹ M-1) inacetonitrile. This shows the separation at preparation of S-BEL andR-BEL respectively by our discovery.

Inhibition of α-Chymotrypsin by BEL—The kinetics of α-chymotrrpsininactivation by (R)-, (S)-, and rac-BEL were performed similar tomethods previously described (36). The concentration of α-chymotrypsin(MW=25,000) was determined using an A^(1%)=20 (1 cm pathlength) at 280nm (37). Briefly, one milliliter of α-chymotrypsin (2 μM) was incubatedwith up to a 5-fold molar excess of (R)-,(S)-, or rac-BEL dissolved inacetonitrile or vehicle alone for 5 mm at 22° C. in 0.1 M sodiumphosphate buffer, pH 7.2. A-10 μl aliquot of the reaction was diluted1000-fold in 10 mL of 0.1 sodium phosphate buffer, pH 7.2 containing 100mM hydrazine to deacylate transiently-inactive BEL-chymotrypsincomplexes. Following incubation at 22° C. for 1 hr, chymotrypsinactivity in each diluted sample was measured utilizing a SPECTRAmaxGeminiXS microplate spectrofluorometer withN-succinyl-Ala-Ala-Pro-Phe-7-amidomethylcoumarin (50 μM) as substratefor 1 mm at 25° C. Excitation and emission wavelengths were 380 nm and460 nm, respectively.

Assay of iPLA₂β and iPLA₂γ Inhibition by BEL—Purified recombinant iPLA₂βor Sf9 cell membranes containing recombinant iPLA₂γ were incubated with(R) BEL, (S) BEL, racemic BEL, or ethanol vehicle for 3 mm at 22° C. inthe presence of 100 mM Tris-HCl (pH 7.0) and 4 mM EGTA (for iPLA₂β) or100 mM Tris-acetate (pH 8.0) and 4 mM EGTA (for iPLA₂γ). Theconcentration of BEL used for each test ranged from 0 to 16 μM.L-α-1-palmitoyl-2-[1¹⁴C]-arachidonyl-phosphatidylcholine (5 μM finalconcentration) or L-α-1-palmitoyl-2-[1-¹⁴C]-oleoyl-phosphatidylcholine(5 μM final concentration) in ethanol was then added to each sample andincubated at 37° C. for 2 min. Reactions were terminated by extractionof radiolabeled products into butanol, and reactants and products wereseparated by thin layer chromatography using Whatman LK6D 60 Å SilicaGel plates with petroleum ether/ethyl ether/acetic acid (70:30:1) as themobile phase. Regions corresponding to the migration of a fatty acidstandard visualized by iodine staining were scraped into vials andradioactivity was quantified by scintillation spectrometry.

Quantification of [³H]-Arachidonic Acid Liberation from A-10Phospholipids—Rat aortic smooth muscle A-10 cells, cultured in 60 mmdishes (2.5×10⁵ cells/dish), were radiolabeled with 0.5 μCi[³H]-arachidonic acid per dish as previously described (29). Cells werewashed once with DMEM containing 0.25% fatty acid free bovine serumalbumin followed by two washes with DMEM alone. Cells were thenincubated with the indicated concentrations of (R)-, (S)-, rac-BEL, orethanol vehicle (0.1% final concentration) in DMEM for 20 min. Thismedia was removed and the cells were then incubated with DMEM containing10% heat-inactivated fetal bovine serum in the absence or presence of 1μM AVP. In some tests, PMA (1 μM) or DOG (10 μM) was added to the mediumcontaining AVP. After 5 mm, 1 ml of this medium was removed, lipids wereextracted into 2 ml of chloroform/methanol/acetic acid (25:24:1 v/v)(38), and the remaining cells were scraped into 1 ml of deionized waterprior to lipid extraction as described above. The chloroform layer wasevaporated under nitrogen and the extracted lipids were separated bythin layer chromatography (petroleum ether/ethyl ether/glacial aceticacid 70:30:1). Regions containing fatty acids and phospholipids werescraped into vials and radioactivity was quantified by liquidscintillation spectrometry.

Measurement of Cytosolic and Membrane-bound PhosphatidatePhosphohydrolase Activities—A-10 cells were grown to confluency, washedtwice in ice-cold phosphate-buffered saline, and harvested in lysisbuffer (50 mM Tris-HCl, pH 7.4 containing 0.25 M sucrose and 0.2 mMDTT). After brief sonication utilizing a Vibra-Cell VC4O sonicator (5×1s pulses at 30% power), the lysed cell suspension was centrifuged at100,000×g for 1 hr to separate cytosolic and membrane fractions. In sometests, A-10 cells were washed and pretreated with BEL or ethanol vehicle(as described above for tests examining [³H]-AA liberation) beforeisolation of cell homogenates. Each fraction (40 μL) was pre-incubatedwith BEL (up to 100 μM) or ethanol vehicle at 22° C. for 5 mm in thepresence of 50 mM Tris-HCl, pH 7.2 containing 10 mM β-mercaptoethanol, 2mM MgCl₂, and 1 mM EGTA (90 μL final volume). Dipaimitoyl phosphatidicacid (100 μM final concentration containing 0.05 μCi L-α-dipalmitoyl[glycerol-¹⁴C(U)]-phosphatidic acid per reaction in the presence of 1 mMTriton X-100) was added to each reaction and incubated at 37° C. for5-10 mm. Reactions were terminated with 900 μL of 5% acetic acid andextracted into chloroform by the method of Bligh and Dyer (38) prior toseparating dipaliitoyl glycerol by TLC utilizing chloroform: methanol:water (65:35:2) as the mobile phase prior to quantification byscintillation spectrometry.

Determination of Phosphorylated MAPK—Confluent A-10 cells in 10 cmdishes were incubated overnight in the presence of DMEM containing 1%fetal bovine serum to reduce background phosphorylation of ERK1 andERK2. Cells were washed twice with DMEM without serum and pre-incubatedwith 5 μM (R)-BEL, (S)-BEL, rac-BEL, or ethanol vehicle in DMEM withoutserum for 15 mm at 37° C. This media was then removed and the cells wereincubated with 1 μM AVP for 5 mm, at 37° C. After washing once withice-cold phosphate-buffered saline (PBS), cells were scraped into RIPAbuffer (PBS, pH 7.4 containing 1% Igepal CA-630, 0.5% sodiumdeoxycholate, 0.1% SDS, 0.5 mM 4-(2-aminoethyl) benzenesulfonylfluoride,10 μg/ml aprotinin, and 1 mM sodium orthovanadate), incubated on ice for30 mm, and then centrifuged at 10,000×g for 10 mm. The proteinconcentrations of the sample supernatants were determined utilizing thebicinchoninic acid (BCA) assay (Pierce) with bovine serum albumin (BSA)as a standard. Samples were electrophoresed according to the method ofLaemmli (39) and transferred to a PVDF membrane by electroelution in 10mM CAPS (pH 11) for ECL Western analysis. After blocking withTris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% non-fat drymilk for 2 hrs, primary rabbit polyclonal antibodies againstphosphorylated (pTEpY) and dephosphorylated MAPK diluted 1:5000 in PBScontaining 5% BSA were incubated with the blot for 1 hr. After washingwith TBS-T, the blots were incubated with ice-cold PBS containing 0.25%glutaraldehyde for 15 mm as previously described (40), washed, andincubated with a protein A-peroxidase conjugate diluted (1:5000) inTBS-T containing 5% BSA for 1 hr. bimunoreactive bands were visualizedby ECL as described by the manufacturer (Amersham Pharmacia).

Determination of PKC Translocation—Confluent A-10 cells in 10 cm disheswere washed twice with DMEM without serum, followed by incubation witheither 5 μM (S)-BEL or ethanol vehicle in DMEM for 15 min at 37° C. Thismedia was then removed and DMEM with or without 1 μM AVP was incubatedwith the cells for 5 min. After washing with ice-cold PBS, the cellswere collected by scraping into 20 mM Tris-HCl, pH 7.4 containing 0.33 Msucrose, 5 mM EDTA, 0.5 mM 4(2-aminoethyl)benzenesulfonylfluoride, and 5μg/ml leupeptin and were lysed by three cycles of flash freezing withliquid nitrogen and thawing. Each sample was then further homogenizedutilizing a teflon homogenizer before isolating the low speed pellet(1,000×g), membrane (100,000×g pellet), and cytosol (100,000×gsupernatant) fractions. The protein concentrations of the fractions weredetermined utilizing the bicinchoninic acid (BCA) assay (Pierce) withBSA as a standard. Samples were electrophoresed and subjected to ECLWestern analysis utilizing rabbit polyclonal antibodies against PKCα andPKCε as described above for MAPK phosphorylation. For blots incubatedwith mouse monoclonal antibodies against PKCδ and PKCι., an anti-mouseIgG (Fab specific)-peroxidase conjugate was utilized in place of theprotein A-peroxidase conjugate.

Results

Separation of BEL Enantiomers—Since BEL contains a chiral center at theC-2 position (FIG. 2), (R)-BEL and (S)-BEL might have differentpotencies and/or selectivities for iPLA₂β and iPLA₂γ so that individualenantiomers of BEL could be exploited to identify the roles of iPLA₂βand iPLA₂γ in agonist-stimulated AA release in intact cells.Accordingly, a chiral HPLC column was used to separate (R)-BEL and(S)-BEL from rac-BEL (FIG. 2). Separation of the BEL enantiomers underthe conditions employed resulted in resolution of two major UV-absorbingpeaks with an RT difference of approximately 2 mm. The first peak elutedat 18.8 mm (Peak A) while the second peak eluted at 20.5 mm (Peak B)(Relative retention time (R_(RT)) Peak A/Peak B=0.917) (FIG. 2).Integration of the areas of each peak were identical (within 1%),suggesting that the enantiomers of BEL had been separated. Proton NMRdata demonstrated peaks with the anticipated chemical shifts andcoupling constants as has previously been published (data not shown)(41). Moreover, electrospray ionization mass spectrometric analysis ofthe material in each peak revealed the presence of a lithiated doubletat 323.3 and 325.3 Daltons (consistent with the presence of a bromineatom) which was of equal intensity for each moiety. Re-injection of PeakA or Peak B onto the chiral column demonstrated that each purifiedmoiety eluted at its previous retention time with negligible amounts ofcontaminating material demonstrating that, as expected, no equilibrationhad occurred. Finally, both peaks coeluted utilizing a non-chiral C18HPLC column (data not shown).

Identification of the Absolute Chirality of the BEL Enantiomers—Todetermine the absolute chirality of the resolved BEL enantiomers,synthetic enantiomers of(F)-6-(iodomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one(αNpI6) of known chirality were chromatographed separately and togetheron the chiral stationary phase. Under these conditions, (S)-αNpI6(R_(T)=19.5 min) eluted prior to (R)-αNpI6 (R_(T)=21.4 min) withapproximately the same degree of separation (R_(RT)=0.911) as the BELenantiomers. Chymotrypsin has previously been identified as a suitabletarget for aromatic haloenol lactones resulting in its mechanism-basedinhibition as detailed by careful kinetic analyses by Katzenellenbogen(41-44). In prior studies, (R)-BEL was determined to be a more efficientinhibitor of chymotrypsin than (S)-BEL in comparison to its chiralcounterpart (36). To further substantiate the absolute stereochemistryof the BEL enantiomers resolved by chiral HPLC and to confirm theability of the resolved enantiomers to selectively inhibit chymotrypsinactivity, increasing concentrations of (R)-BEL, (S)-BEL, or rac-BEL wereincubated with chymotrypsin, and diluted in buffer as described in“Experimental Procedures”. Activity assays were performed using thefluorogenic chymotrypsin substrateN-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin as described in“Experimental Procedures”. Under the conditions employed, (R)-BELstoichiometrically and irreversibly inhibited chymotrypsin while (S)-BELwas considerably less potent (FIG. 3). These results confirm that bothpeaks are distinct enantiomers of BEL and substantiate, by independentcriteria, the assigned absolute stereochemistry of the peaks elutingfrom the chiral HPLC column.

Enantiomeric Selective inhibition of iPLA₂β and iPLA₂γ—Previousexperiments with iPLA₂β and iPLA₂γ have established that iPLA₂β andiPLA₂γ are inhibited by racemic BEL with an IC₅₀ range of 200 nM foriPLA₂β and about 3 μM for iPLA₂γ (24, 31). Accordingly, we next examinedthe ability of the resolved enantiomers of BEL to inhibit iPLA₂β andiPLA₂γ. For these experiments, iPLA₂β or iPLA₂γ was pre-incubated with(R)-BEL, (S)-BEL, rac-BEL, or ethanol vehicle alone for 3 mm followed bymeasurement of remaining enzymatic activity utilizing a radiolabeledphospholipid substrate. Remarkably, (S)-BEL selectively inhibited iPLA₂β10-fold more potently than (R)-BEL (FIG. 4A). In stark contrast, (R)-BELselectively inhibited iPLA₂γ approximately 10-fold more potently than(5)-BEL (FIG. 4B). As anticipated, the inhibitory potency of racemic BELwas intermediate of that of (R)-BEL and (S)-BEL. Thus, (5)-BEL is a morepotent inhibitor of iPLA₂β while (R)-BEL is a more potent inhibitor ofiPLA₂γ.

Identification of iPLA2β and not iPLA2γ as the Mediator of AVP-inducedAA Release in A-10 Cells—Upon stimulation with AVP, A-10 smooth musclecells rapidly release a relatively large percentage (about 5%) of theiresterified arachidonic acid (10). Prior studies have demonstrated thatpretreatment of the cells with 2 to 5 μM BEL inhibits≈60 to 80%,respectively, of AVP-inducible arachidonic acid release (10, 29).Moreover, the absence of extracellular calcium ion (incubationsperformed in the presence of EGTA in the media) or the presence ofintracellular calcium ion chelators (e.g. BAPTA) does not effectAVP-induced AA release in A-10 cells (29). Since combined incubationswith EGTA and BAPTA completely ablated FURA-2 observable increases inintracellular calcium ions, these results further implicated theinvolvement of a calcium-independent phospholipase A₂ in this process.However, since both iPLA₂β and the newly identified iPLA₂γ are bothinhibited by rac-BEL (and are calcium-independent), the identity of theiPLA₂ mediating AA release in AVP-stimulated A-10 cells was unknown. Toaddress this issue, we exploited the selectivity of (S)-BEL and (R)-BEL,for inhibition of iPLA₂β and iPLA₂γ, respectively, to determine the typeof iPLA₂ catalyzing AVP-induced release of [³H]-armchidonic acid fromA-10 cells. As previously demonstrated, A-10 cells stimulated with AVPresults in a substantial increase in the amount of nonesterified[³H]-arachidonic acid relative to control cells incubated with vehiclealone (FIG. 5). This AVP-induced increase in non-esterified[³H]-arachidonic acid is significantly reduced in the presence of lowconcentrations of rac-BEL (1 and 5 μM) (FIG. 5). Importantly, (S)-BEL (1μM) substantially inhibited AA release (50% inhibition) and 5 μM (S)-BELcompletely attenuated AVP-induced AA release. In sharp contrast, (R)-BELis virtually ineffective (about 10% inhibition at 5 μM BEL) ininhibiting AVP-induced AA release from A-10 cells under similarconditions (FIG. 5). Thus, iPLA₂β, and not iPLA₂γ, is the likelymediator of AA release in this system.

Confirmation of the Lack of Effects of BEL on Processes TypicallyAssociated with cPLA2α Activation—Activation of cPLA2α in most systemsdepends on the concomitant activation of MAPK, PKC, and increases inintracellular [Ca²⁺] (45-47). In prior studies we demonstrated that BELdoes not inhibit AVP-induced increases in [Ca²⁺]₁, and that ablation ofchanges in [Ca²⁺] by BAPTA does not attenuate AA release (29). Recently,Dennis and coworkers have suggested that cytosolic phosphatidatephosphohydrolase (PAP-1) in some cell types may be a target for BEL andthat the resulting inhibition of PAP-1 would result in diminished levelsof diacylglycerol produced from phosphatidic acid, thereby attenuatingPKC activation precluding cPLA₂α activation and AA release (33, 34). Toaddress this possibility, we first examined the effects of rac-BEL onA-10 cell PAP activities in cytosol and membrane fractions (FIG. 6A) aswell as in intact cells (FIG. 6B). These tests consistently demonstratedthe lack of any effect of BEL on either the cytosolic (PAP-1) or themembrane-bound (PAP-2) forms of A-10 cell phosphatidate phosphohydrolaseat concentrations up to 200 μM BEL (FIG. 6A). Furthermore, homogenatesfrom intact A-10 cells previously exposed to up to 100 μM racemic BELdid not inhibit total phosphatidate phosphohydrolase activity incomparison to ethanol-treated controls (FIG. 6B). Next, we examinedwhether activation of PKC by exogenous addition of eitherphorbol-12-myristate-13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol(DOG) could rescue AA release after BEL pretreatment. Neither PMA or DOGcould restore the ability of (S)-BEL treated A-10 cells to releasearachidonic acid, thereby demonstrating that BEL is likely inhibitingarachidonic acid release in a manner which is no longer responsive toPKC activation (i.e. irreversible covalent modification of iPLA₂) (FIG.7). A-10 cells contain at least four PKC isoforms, PKC_(α), PKC_(δ),PKC_(ε), and PKC₁₂, by Western blot analysis (FIG. 8), however no bandscorresponding to PKC_(βI), PKC_(βII), PKC_(γ), PKC_(η), or PKC_(ζ) couldbe visualized previously. Accordingly, we treated A-10 cells with AVPand determined if (S)-BEL could inhibit PKC translocation to themembrane fraction. Stimulation of A-10 cells with AVP causestranslocation of PKC_(δ) and PKC_(ε) from the cytosol to the membranefraction, but neither PKC_(α) nor PKC_(ι) undergo AVP-inducedtranslocation in A-10 cells (FIG. 8). Pretreatment of A-10 cells with 5μM (S)-BEL, which causes almost complete inhibition of AA release, doesnot effect the translocation of either PKC_(δ) or PKC_(ε) (FIG. 8).Finally, AVP-induced phosphorylation of ERK2 is not effected by thepresence of 5 μM (R)-, (5)-, or rac-BEL (FIG. 9). Collectively, theseresults demonstrate that 1) neither PAP-1 nor PAP-2 is a target for BELin A-10 smooth muscle cells; 2) BEL does not appreciably effect PKC_(δ)and PKC_(ε) translocation or MAPK phosphorylation in A-10 cells; and 3)iPLA₂β is likely responsible for the large majority of arachidonic acidrelease from A-10 cells.

Discussion—Genetic approaches have now demonstrated the presence of twotypes of iPLA₂ activities present in the human genome (iPLA₂β andiPLA₂γ) which are both inhibited by rac-BEL (24, 31, 32). Accordingly,all prior tests demonstrating inhibition of arachidonic acid release byrac-BEL cannot discriminate between hydrolysis catalyzed by iPLA₂β orthat mediated by iPLA₂α. Virtually nothing is known about the regulationof iPLA₂γ or its potential role in agonist-stimulated eicosanoidrelease. In this work, we: 1) resolve rac-BEL by chiral HPLC; 2) assignthe absolute stereochemistry of the resolved enantiomers by twoindependent techniques; 3) demonstrate a 10-fold selectivity of (S)-BELfor inhibition of iPLA₂β and a 10-fold selectivity of (R)-BEL forinhibition of iPLA2γ; 4) demonstrate that (S)-BEL inhibits that vastmajority of AVP-induced AA liberation in A-10 cells while (R)-BEL doesnot; 5) provide evidence that BEL-mediated inhibition of AA release inA-10 cells is not mediated through inhibition of either membrane-boundor cytosolic phosphatidate phosphohydrolases; and 6) demonstrate thattreatment of A-10 cells with (S)-BEL does not attenuate PKCtranslocation or MAPK activation after AVP stimulation. Collectively,these results, in combination with work (see below) demonstrates thatAVP-stimulated AA release in A-10 cells is likely mediated by iPLA₂β andnot iPLA2γ, cPLA₂α, or chymotrypsin-like proteases.

The utilization of chiral pharmacologic agents instead of racemicmixtures has increasingly been appreciated to enhance the potency ofinhibitors toward targeted processes and markedly reduce toxicity and“non-specific inhibition” mediated by interactions with non-targetedsystems. Since enzymes possess multiple chiral centers, the interactionbetween a chiral inhibitor and one or more optically active centers ator near the enzyme active site results in diastereotopic interactionswhich possess different physical properties and spatial relationshipsfor each diastereotopic pair. In the case of mechanism-based inhibitorssuch as BEL, these diastereotopic interactions are anticipated to: 1)alter binding; 2) modify the rate of formation of the acyl-enzymeintermediate; and 3) alter the covalent trapping of the halomethylketone in the acyl enzyme by nucleophiles at or near the active site. Inthis study, we have exploited diastereotopic interactions betweenindividual enantiomers of BEL and the known mammalian iPLA₂s (i.e.iPLA₂β and iPLA₂γ) to achieve a remarkable specificity for inhibition ofiPLA₂β by (S)-BEL and iPLA₂γ by (R)-BEL, respectively. Moreover, wedemonstrated that proteases with similar stereochemical relationships aschymotrypsin are more likely to be inhibited by (R)-BEL than (S)-BEL,thereby further increasing the utility of mechanism-based inhibition togain insight into the types of phospholipases A2 mediating AA release inmammalian cells.

With any pharmacologic compound, unanticipated effects on non-targetedsystems may occur with increasing likelihood at high concentrations ofinhibitor. Mammalian cells have in excess of 30,000 genes which aftersplicing and post-translational modification give rise to well over 10⁵and perhaps as many as 10⁶ different chemical moieties. Of course, it isimpossible to test every compound with each of these chemical moietiesin each different microenvironment in the cell in which pharmacologicagents might interact. Indeed, at high enough concentrations in aqueoussystems, virtually any low molecular weight organic compound willinteract with a diverse array of proteins due to hydrophobic effectsalone. That is precisely why it is important to examine biologic effectselicited by pharmacologic agents at or near their effective inhibitoryconcentrations in intact cells as was determined in isolated purifiedsystems. In the case of BEL, some investigators have employed 50-100 μMBEL which exceeds the effective inhibitory concentration of BEL for theknown mammalian iPLA₂s by two orders of magnitude. Accordingly, thesetests must be interpreted with caution given the IC₅₀ of rac-BEL foriPLA₂β and iPLA₂γ is in the 0.5-3 μM range. Moreover, the mere exposureof cells to high concentrations of organic compounds (50-100 μM) islikely to perturb the fragile order of the membrane microenvironmentand, in the case of investigating membrane-related phenomena, may haveeffects which are independent of interactions with targeted enzymesystems alone. Indeed, we have observed cell death employing 100 μM BELwhich is almost certainly independent of the effects of BEL on targetedprocesses.

Dennis and co-workers have contended that high concentrations of rac-BEL(≈25 μM) effectively inhibit magnesium-dependent cytosolic phosphatidatephosphohydrolase (PAP-1) in mouse P388D1 macrophages (33) and humanamnionic WISH cells (34). The authors argue that inhibition of PAP-1would be expected to cause a deficiency in DAG thus blunting PKCactivation and possibly activation of cPLA2a by MAPK. However, BEL hasbeen subsequently shown not to affect PMA-induced translocation of PKC(or PKC catalytic activity) in P388D1 macrophages (15) or MAPKphosphorylation in WISH cells (34), rat neutrophils (48), and A-10 cells(this application). It should be mentioned that in their investigations,Balsinde et al. did not observe any effect of BEL on PAP-2 (33), thephosphatidate phosphohydrolase isoform which is believed to be involvedin lipid signal transduction pathways (49-51). This absence of PAP-2inhibition by BEL has since been observed in McA-RH7777 rat hepatomacells (17), pancreatic islet cells (52), and A-10 smooth muscle cells(this application). Furthermore, cytosolic PAP-1 activity in McA-RH7777cells is not significantly inhibited by 100 μM BEL (17). A secondpossible effect of inhibited PAP-catalyzed DAG production, as describedby Balboa et al. (34), is that the phospholipid substrate will be in asub-optimal environment since DAG has been demonstrated to altermembrane bilayers by creating more distance between phospholipidheadgroups, thereby making the phospholipid ester linkages moresusceptible to PLA₂-mediated hydrolysis. However, we have found thatrac-BEL does not inhibit release of IP₂ or IP₃ in A-10 cells (10) andAkiba et al. (15) have found that BEL (up to 5 μM) does notsignificantly effect levels of diacylglycerol or phosphatidic acidformed in P388D1 macrophages upon stimulation with zymosan.

The identification of chiral specificity of individual enantiomers ofBEL to inhibit the known mammalian iPLA₂s extends the utility ofmechanism-based inhibitors in the study of agonist-mediated AA release.The tests described herein allow assignment of AVP-induced AA release inA-10 cells to iPLA₂β and not iPLA₂γ. The inhibition of AA release by(S)-BEL and not (R)-BEL excludes participation of chymotrypsin orchymotrypsin-like proteases in these processes. Finally, the utilizationof chiral mechanism-based inhibitors reduces potential “non-specific”complications through comparisons of the effects of specific opticalantipodes on the observed end points (i.e. AA release) with their invitro potency in purified systems. Assignation of specific enzymes aseffectors of AA release requires detailed concurrent consideration ofbiochemical, pharmacologic, and genetic perturbations on the observedprocess. In the case of AVP-induced AA release from A-10 cells, wehave: 1) demonstrated that concentrations of (S)-BEL near the IC₅₀ foriPLA₂β dramatically attenuate AA release in intact A-10 cells while(R)-BEL does not (this application); 2) demonstrated that BEL-mediatedinhibition of AA release occurs in the presence of normal increases in[Ca²⁺]1 (29) and cannot be rescued by exogenous activation of PKC byPIVIA or DOG (this application); 3) that AVP-mediated AA release in A-10cells is not affected by removal of calcium ions from the external mediaor by effective buffering internal calcium concentration by BAPTA-AM(entry of extracellular calcium and increases in [Ca²⁺]1 are eachthought to be necessary for cPLA₂α activation (53-55)); and 4) that manyother enzymes thought to be necessary or associated with AA release arenot inhibited by the concentrations of BEL employed. For example,enzymes which participate in signal transduction cascades which areknown not to be inhibited by the concentrations of BEL employed include:phosphatidylinositol-specific phospholipase C (10), phospholipase D(17), protein kinase A (56), and channels which mediate Ca²⁺ releasefrom intracellular stores (10). Of course, as with any other process, wecan not rule out the involvement of as yet undiscovered phospholipasesor activation cascades. However, we state that in the presence of PKCactivation, MAPK activation, and increases in [Ca²⁺], (processestypically deemed necessary for cPLA2a-mediated AA release), arachidonicacid release is still inhibited by (S)-BEL with a dose response profileand chiral selectivity which closely corresponds to iPLA₂β.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1—shows the calcium-independent phospholipase A₂ (iPLA₂) genefamily and sequence alignment of iPLA₂ nucleotide and lipase consensusmotifs. Members of the iPLA₂ gene family (α, β, and γ) are alignedaccording to their nucleotide-binding motifs (boxed diagonal bars) and,lipase consensus sites (filled bars). Calcium-independent phospholipaseA₂β contains eight ankyrin repeat domains (gray bars) and acalmodulin-binding domain (CaM) near the C-terminus (boxed horizontalbars).

FIG. 2 shows separation of BEL enantiomers by chiral HPLC. A Chirex3,5-dinitrobenzoyl-(R)-phenylglycine chiral HPLC column (4.6 mm×25 cm)was equilibrated with hexane/dichloroethane/ethanol (150/115/1) at aflow rate of 2 ml/min. Racemic BEL (10 nmol) was injected onto thecolumn as feed and the UV absorbance (280 nm) was recorded for the timeindicated. The elution time for the (S) enantiomer (Peak A) was 18.8 mmand that for the (R) enantiomer (Peak B) was 20.5 mm. The chemicalstructures of (R)- and (S)-BEL are as indicated above the chromatogram.

FIG. 3 shows inhibition of α-chymotrypsin by racemic, (R)-, and (S)-BEL.Chymotrypsin (2 μM) in 0.1 M sodium phosphate buffer, pH 7.2 wasincubated with the indicated concentrations of racemic, (R)-, or (S)-BELfor 5 mm. This solution was then diluted 1000-fold in 0.1 M sodiumphosphate buffer, pH 7.2 containing 100 mM hydrazine and incubated for 1hr at 22° C. Chymotrypsin proteolytic activity was measuredspectrofluorometrically usingN-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin (50 μM) assubstrate.

FIG. 4 shows selective inhibition of iPLA₂β and iPLA₂γ by racemic, (R)-,and (S)-BEL. Panel A—Purified recombinant iPLA₂β (1 μg/ml) in 100 mMTris-HCl, pH 7.3 containing 1 mM EGTA was preincubated at 22° C. for 3mm in the presence of the indicated concentrations of (R)-, (S)-, orrac-BEL or ethanol vehicle alone (1% final concentration). Radiolabeledsubstrate (L-α-1-palmitoyl-2-[1-¹⁴C]-arachidonyl-phosphatidylcholine, 5μM final concentration) was then added to each reaction and incubated at37° C. for 2 min. Reactions were terminated by extraction of remainingsubstrate and products into butanol, separation by TLC, andquantification of released radiolabeled fatty acid by scintillationspectrometry as described in Experimental Procedures. Results arerepresentative of four separate tests. Panel B—Washed Sf9 cell membranescontaining recombinant human iPLA₂γ were preincubated for 3 min with theindicated concentrations of (R)-, (S)-, or racemic BEL or ethanolvehicle alone (1% final concentration) in 100 mM Tris-acetate, pH 8.0containing 1 mM EGTA at 22° C. Radiolabeled substrate(L-α-1-palmitoyl-2-[1-¹⁴C]-oleoyl-phosphatidylcholine, 5 μM finalconcentration) was then added to each reaction and incubated at 37° C.for 2 min. Reactions were terminated by extraction of remainingsubstrate and products into butanol, separation by TLC, andquantification of released radiolabeled fatty acid by scintillationspectrometry as described in “Experimental Procedures”. Results arerepresentative of four separate tests.

FIG. 5 shows inhibition of AVP-mediated arachidonic acid liberation inA-10 smooth muscle cells by racemic, (R)-, and (S)-BEL. A-10 cells(2.5×10⁵ cells/dish) were radiolabeled with [³H]-arachidonic acid (0.5μCi/dish) for 20 hrs. After washing to remove unincorporated[³H]-arachidonic acid, cells were then incubated with either 1 μM or 5μM (R)-BEL, (S)-BEL, rac-BEL, or ethanol vehicle (0.1%) in DMEM for 20mm. This media was removed and the cells were then incubated with 3 mlof DMEM containing 10% heat-inactivated fetal bovine serum with orwithout 1 μM AVP. After 5 min, 1 ml of this medium was removed andlipids were extracted into 2 ml of chloroform/methanol/acetic acid(25:24:1). The remaining cells were scraped into 1 ml of deionized waterand total lipids were extracted as described above. The chloroform layerwas evaporated under nitrogen and the extracted lipids were separated byTLC. Regions containing fatty acids and phospholipids were scraped intovials and radioactivity was quantified by liquid scintillationspectrometry. Results are representative of four separate tests.

FIG. 6 shows the inability of racemic BEL to inhibit A-10 smooth musclecell cytosolic (PAP-1) and membrane-bound (PAP-2) phosphatidatephosphohydrolase activities. A-10 cells plated on 100 mm dishes werewashed 2× in ice-cold PBS and harvested in lysis buffer (50 mM Tris-HCl,pH 7.4 containing 0.25 M sucrose and 0.2 mM DTT). After brieflysonicating, cytosolic and membrane fractions were separated byultracentrifigation before measuring phosphatidate phosphohydrolaseactivity. In panel A, cytosolic or membrane fractions were pre-incubatedwith rac-BEL (up to 200 μM) or ethanol vehicle at 22° C. for 5 min inthe presence of 50 mM Tris-HCl, pH 7.2 containing 10 mMβ-mercaptoethanol, 2 mM MgCl₂, and 1 mM EGTA. Dipalmitoyl phosphatidicacid (100 μM final concentration containing 0.05 μCi L-α-dipalmitoyl[glycerol-¹⁴C(U)]-phosphatidic acid per reaction in the presence of 1 mMTriton X-100) was added to each reaction which was then transferred to a37° C. water bath for 5 (membrane fraction) or 10 (cytosolic fraction)mm. Reaction products were terminated with the addition of 5% aceticacid, extracted into chloroform, dried under nitrogen, and resolved byTLC as described in “Experimental Procedures”. Results arerepresentative of three separate tests. In panel B, intact A-10 cellswere washed and pretreated with BEL or ethanol vehicle (as described inFIG. 5) before isolation of the cell homogenate and quantification ofphosphatidate phosphohydrolase activity as described above. Results arerepresentative of three separate tests.

FIG. 7 shows the inability of phorbol-12-myristate-13-acetate and1,2-dioctanoyl-sn-glycerol to reconstitute arachidonic acid liberationin A-10 cells treated with (S)-BEL. A-10 cells were radiolabeled with[³H]-arachidonic acid, washed, and pre-incubated with 5 μM (S)-BEL orethanol vehicle alone for 15 min as described in FIG. 5. DMEM mediacontaining 10% heat-inactivated fetal bovine serum with or without 1 μMAVP was then added to the cells. PMA (1 μM) or DOG (10 μM) were includedin this media as indicated in the figure. After 5 min at 37° C.,[³H]-arachidonic acid released into the media and remaining within thecells (free and incorporated into phospholipids) was extracted intochloroform, separated by TLC, and quantified by liquid scintillationspectrometry as described in “Experimental Procedures”. Results arerepresentative of three separate tests.

FIG. 8 shows that translocation of PKCδ and PKCε in A-10 smooth musclecells is unaffected by pre-treatment with (S)-BEL. A-10 cells wereincubated in the presence of 5 μM (S)-BEL or ethanol vehicle in DMEMwithout serum for 15 min at 37° C., followed by removal of the medium,and incubation in the presence or absence of 1 μM AVP in DMEM for 5 minas indicated in the figure. After lysis of the cells, low speed pellets(LSP) obtained by centrifugation at 1,000×g, membrane (Memb), andcytosolic (Cyto) fractions (obtained by centrifugation at 100,000×g)were electrophoresed by SDS-PAGE (20 μg of protein per lane) andimmunoreactive bands corresponding to the indicated PKC isoforms (α, δ,ε, and ι) were visualized by ECL Western analysis as described in“Experimental Procedures”.

FIG. 9 shows that racemic BEL, (R)-BEL, or (S)-BEL do not inhibitAVP-induced MAPK phosphorylation. A-10 cells were incubated in thepresence of ethanol vehicle or 5 μM rac-BEL, (R)-BEL, or (S)-BEL in DMEMfor 15 min at 37° C. After removal of this media, the cells wereincubated for 5 min in the presence or absence of 1 μM AVP as indicatedin the figure, washed with ice-cold PBS, and then lysed with RIPAbuffer. Extracts from the cells were separated by SDS-PAGE (20 μgprotein per lane), transferred to a PVDF membrane, and analyzed by ECLimmunoblotting for phosphorylated MAPK as described in “ExperimentalProcedures”.

Abbreviations

AVP—Arginine vasopressin

BEL is(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one

Rac-BEL—Racemic(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one

(R)-BEL—(R)(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one

(S)-BEL—(S)(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one

αNpI6—(E)-6-(iodomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one

EGTA—Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

cPLA₂—Cytosolic phospholipase A₂

iPLA₂—Calcium-independent phospholipase A₂

iPLA₂β—Calcium-independent phospholipase A₂β

iPLA₂γ—Calcium-independent phospholipase A₂γ

PAP—Phosphatidate phosphohydrolase

PMA—Phorbol-12-myristate-13-acetate

DOG—1,2-Dioctanoyl-sn-glycerol

R_(RT)—Relative retention time

Rac-BEL=racemic BEL

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While the invention has been described with respect to various specificexamples and embodiments thereof, it is understood that the invention isnot limited thereto and many alternatives, modifications, and variationswill be apparent to those skilled in the art in light of the foregoingdescription. Accordingly, it is intended to embrace all suchalternatives, modifications, and variations as fall within the spiritand broad scope of the invention.

1. A process for preparing S-BEL which comprises passing a racemicmixture of BEL through a chromatographic column packed with an opticalresolution packing material and optically resolving S-BEL from theracemic mixture.
 2. A process in accordance with claim 1 wherein saidpacking material comprises silica.
 3. A process in accordance with claim2 wherein said column comprises 3,5-dinitrobenzoyl-(R)-phenylglycine. 4.The process of claim 3, wherein said separation affords at least one ofthe enantiomers a recovery of greater than or equal to about 60%.
 5. Theprocess of claim 4, wherein said separation affords at least one of theenantiomers a recovery of greater than or equal to about 90%.
 6. Amethod of separating a racemic composition comprising R-BEL and S-Belinto purified R-BEL and S-BEL respectively which comprises passing saidracemic composition through an HPLC chiral separation column whichcomprises a stationary phase and an immobile phase and eluting saidR-BEL and said S-BEL separately from said composition.
 7. A process inaccordance with claim 6 wherein said packing material comprises silica.8. A process in accordance with claim 7 wherein said column comprises3,5-dinitrobenzoyl-(R)-phenylglycine.
 9. The process of claim 8, whereinsaid separation affords at least one of the enantiomers a recovery ofgreater than or equal to about 60%.
 10. The process of claim 9, whereinsaid separation affords at least one of the enantiomers a recovery ofgreater than or equal to about 90%.
 11. A process forchromatographically resolving enantiomerically pure or opticallyenriched BEL from a mixture containing two enantiomers of BEL usingchiral chromatography comprises a liquid mobile phase comprising3,5-dinitrobenzoyl-phenylglycine.
 12. A process in accordance with claim11 wherein said packing material comprises silica.
 13. A process inaccordance with claim 12 wherein said column comprises3,5-dinitrobenzoyl-(R)-phenylglycine.
 14. The process of claim 13,wherein said separation affords at least one of the enantiomers arecovery of greater than or equal to about 60%.
 15. The process of claim14, wherein said separation affords at least one of the enantiomers arecovery of greater than or equal to about 90%.
 16. Chiralchromatographically prepared isolated and purified S-BEL.
 17. Chiralchromatographically prepared isolated and purified R-BEL.
 18. A methodin accordance with claim 1 wherein said method comprises preparativeHPLC.
 19. A method in accordance with claim 18 wherein said methodcomprises chemical separation.
 20. A method in accordance with claim 19wherein said method chemical separation comprises chiral separation. 21.A method in accordance with claim 20 wherein said chiral separationcomprises contacting said compositions with a stationary phase and animmobile phase said immobile phase having a selective retention.
 22. Achiral chromatographically prepared purified composition comprisingR-BEL.
 23. A chiral chromatographically prepared purified compositioncomprising S-BEL.
 24. A method of identifying whether an enzyme ismetabolically active within a cellular environment, which comprisescontacting said cellular environment with at least one of S-BEL andR-BEL and determining the identity of the enzyme based on theinteraction of said enzyme with at least one of S-BEL and R-BEL.
 25. Amethod in accordance with claim 26, wherein said enzyme is a lipase. 26.A method in accordance with claim 27 wherein said identity is determinedby selective inhibition.
 27. A method in accordance with claim 28wherein said lipase is contacted with S-BEL.
 28. A method in accordancewith claim 29 wherein said lipase is contacted with R-BEL.
 29. A methodof identifying whether a lipase enzyme is metabolically active within acellular environment, which comprises contacting said cellularenvironment with at least one of S-BEL and R-BEL and determining theidentify of the lipase based on the interaction of said lipase withS-BEL.
 30. A method of identifying whether a lipase enzyme ismetabolically active within a cellular environment, which comprisescontacting said cellular environment with at least one of S-BEL andR-BEL and determining the identity of the lipase based on theinteraction of said lipase with R-BEL.
 31. A method in accordance withclaim 30, wherein said identity of the lipase is based on selectivity.32. A diagnostic method for determining the metabolic activity of alipase within a cellular environment, which comprises contacting saidcellular environment with at least one of S-BEL and R-BEL anddetermining the identity of the lipase based on the interaction of saidlipase with at least one of S-BEL and R-BEL.
 33. A diagnostic kit fordetermining the metabolic activity of a lipase within a cellularenvironment, which comprises providing said cellular environmentcomprising a lipase for which it is desired to determine the metabolicactivity, contacting said cellular environment with at least one ofS-BEL and R-BEL and determining the identify and/or activity of thelipase based on the interaction of said lipase with at least one ofS-BEL and R-BEL.
 34. A method of differentially inhibiting iPLA₂γ andiPLA₂β by contacting the same with S-BEL, observing selectivity of S-BELtoward iPLA₂γ or iPLA₂β and determining that said iPLA₂β has beeninhibited.
 35. A method of inactivating a lipase enzyme which ismetabolically active within a cellular environment, which comprisescontacting said cellular environment with at least one of S-BEL andR-BEL thereby rendering the lipase inactive.
 36. A method in accordancewith claim 35 wherein said cellular environment comprises at least oneof a cell, biological entity and living organism.
 37. A method inaccordance with claim 36 wherein said lipase is an iPLA₂ lipase.
 38. Amethod of selectively inhibiting iPLA₂γ in a composition by effectivelycontacting the same with an effective inhibiting amount of S-BEL.
 39. Amethod for pharmacologically distinguishing iPLA₂γ from iPLA₂β whichcomprises contacting a candidate iPLA₂ with S-BEL and if the selectivityis high of S-BEL to the candidate iPLA₂, then determining that the iPLA₂is iPLA₂γ. As used herein, the term “candidate” includes members of theiPLA₂ family.